The Sirtuins are a conserved family of NAD+-dependent protein deacetylases that function in a wide variety of cellular processes related to aging and age-associated disease. Due to their dependence on NAD+, sirtuin activity is closely linked to the metabolic and energy status of the cell, and therefore, has been proposed to help mediate the beneficial effects of caloric restriction. As a result, there is significant interest i characterizing additional mechanisms by which sirtuin activity is modulated, whether it is through protein-protein interactions, post-translational modifications, or via small molecule agonists and inhibitors. Sir2 from the budding yeast, Saccharomyces cerevisiae, is the founding family member and has been extensively studied for its role in transcriptional silencing at telomeres, the silent mating-type loci, and the rDNA. It has also served as an outstanding model system for interrogating Sir2 function in lifespan regulation. In preliminary experiments we have identified a novel RNA binding activity for yeast Sir2 that requires an intact zinc ribbon domain. The RNA interaction also stimulates the HDAC activity of Sir2 in vitro, suggesting that RNA or possibly other nucleic acid species may positively regulate Sir2 in vivo. Recent ChIP-Seq data has revealed that Sir2 surprisingly associates with active genes, in addition to the traditional silenced loci, thus supporting the hypothesis that Sir2 and RNA may functionally interact. In this exploratory project we propose two specific aims. The first is designed to identify RNA sequences that Sir2 interacts with in vivo, and to determine if there is any sequence specificity to the interaction. In the second specific aim, we propose to quantitatively characterize the binding affinity between Sir2 and RNA, and to determine the level of conservation with sirtuins from other species such as human SIRT1, the closest ortholog to yeast Sir2. To complement the binding experiments, the mechanism by which RNA stimulates HDAC activity will be investigated to determine whether RNA impacts catalysis (Vmax) or substrate binding (Km). These exploratory and developmental studies will define the basic mechanism and specificity of the functional Sir2-RNA interaction and set us up for more detailed functional and structure-function studies in the future.